1. Field of the Invention (Technical Field)
The present invention relates to fluorescence sensing systems employing excited-state lifetime detection for use, for example, in chemical and biological sensors.
2. Background Art
Note that the following discussion refers to a number of publications by author(s) and year of publication, and that due to recent publication dates certain publications are not to be considered as prior art vis-a-vis the present invention. Discussion of such publications herein is given for more complete background and is not to be construed as an admission that such publications are prior art for patentability determination purposes.
Designers of chemical and biological sensors often choose fluorescence as their detection modality because it offers high sensitivity and a large choice of fluorescence sensor transduction techniques. M. Sekar, et al., J. Am. Chem. Soc. 121, 5135-5141 (1999). Sensor systems that utilize some form of excited-state lifetime detection have several advantages over those that use simple intensity detection. Most of these benefits are in the form of reduced sensitivity to undesirable effects, including changes in excitation light intensity and in dye concentration, fluorophore bleaching, and sample turbidity. E. Rabinovich, et al., Rev. Sci. Instrum. 71, 522-529 (2000); H. Szmancinski, et al., Sens. Actuators B 29, 16-24 (1995).
There are two well-developed classes of excited-state lifetime detection techniques. Time-domain methods rely on direct measurements of fluorescence intensity decay. Frequency-domain methods (e.g., modulation spectroscopy) measure the effects of finite fluorescence lifetimes on the sinusoidal intensity modulation of fluorescence emission, typically the effects on modulation depth and (or) modulation phase shifts. H. Szmancinski, et al., supra.
The present invention is based on modulation spectroscopy. Changes in fluorescence lifetime alter the frequency of auto-oscillations in a closed-loop optoelectronic circuit. The oscillation exists as a radio frequency (RF) sinusoidal modulation of the fluorescence excitation and emission intensity. This technique is relatively straightforward to implement and is inexpensive. It has high sensitivity over a broad range of lifetimes because a wide range of frequencies may be measured precisely.
The present invention is of a fluorescence average excited-state lifetime sensor comprising: a fluorescence excitation light source; light-directing apparatus directing light from the light source to a sample; light-receiving apparatus receiving fluorescence light generated by the sample; and a narrow-band resonance amplifier providing gain necessary to support self-oscillations in an opto-electronic loop comprising the light source, the sample, the light-directing apparatus, the light-receiving apparatus, and the resonance amplifier. In the preferred embodiment, light from the light source and the fluorescence light have different wavelengths and each carry a radio frequency intensity modulation in the loop. The light source can be a light emitting diode (e.g., blue). The light-receiving apparatus preferably comprises a long-wavelength-pass optical filter to prevent reception of light from the light source. The light-directing apparatus preferably directs light to a plurality of fluorophores, and the light-receiving apparatus receives light with radio frequency intensity modulation of identical frequency as that of the light source but phase shifted. The light-receiving apparatus preferably comprises a photomultiplier tube, with the light-receiving apparatus connected to an input of the resonance amplifier. The amplifier preferably comprises a resonance radio frequency amplifier, preferably with the central frequency of the amplifier and lengths of the light-directing and light-receiving apparatus being such that the RF modulation frequency multiplied by the excited-state fluorescence lifetime of fluorophores in the sample is approximately one. The sensor preferably comprises a frequency counter receiving a signal between the amplifier and the light source, as well as a second light source and a photodetector receiving output from the second light source and providing input to the frequency counter. The light source can be a diode laser (e.g., blue or red). The resonance amplifier preferably prevents higher order oscillations. An alternative embodiment adds an electronic phase shifter within the opto-electronic loop and also a second opto-electronic loop comprising the electronic phase shifter, a second apparatus for receiving fluorescence light generated by the sample, and a phase detector. In that embodiment, light from the light source is used as a light carrier of radio frequency intensity modulation in the first opto-electronic loop, and the fluorescence light is used as a light carrier of radio frequency intensity modulation in the second opto-electronic loop. The sensor preferably has a sub-picosecond resolution for changes in average excited-state lifetime of the sample. The sensor can measure changes of chemical environment when the sample exhibits changes of fluorescence average excited-state lifetime in response to changes of the chemical environment, changes of physical environment when the sample exhibits changes of fluorescence average excited-state lifetime in response to changes of the physical environment, changes of concentration of one or more chemical species when the sample exhibits changes of fluorescence average excited-state lifetime in response to changes of concentration of the chemical species, and changes of concentration of one or more biological species when the sample exhibits changes of fluorescence average excited-state lifetime in response to changes of concentration of the biological species.
The invention is also of a fluorescence average absolute lifetime sensor comprising: a fluorescence excitation light source; light-directing apparatus directing light from the light source to a sample; light-receiving apparatus receiving fluorescence light generated by the sample; an electronic phase shifter; and a narrow-band resonance amplifier providing gain necessary to support self-oscillations in an opto-electronic loop comprising the light source, the sample, the light-directing apparatus, the light-receiving apparatus, the phase shifter, and the resonance amplifier. In the preferred embodiment, light from the light source and the fluorescence light have different wavelengths and each carry a radio frequency intensity modulation in the loop. The amplifier is preferably a resonance radio frequency amplifier, with the central frequency of the amplifier and lengths of the light-directing and light-receiving apparatus being such that an RF modulation frequency multiplied by an excited-state fluorescence lifetime of fluorophores in the sample is not approximately one. The light-directing apparatus preferably directs light to a plurality of fluorophores, with the light-receiving apparatus receiving light with radio frequency intensity modulation of identical frequency as that of the light source but phase shifted.
The invention is additionally of a fluorescence average excited-state lifetime sensing method comprising: exciting a fluorescence excitation light source; directing light from the light source to a sample; receiving fluorescence light generated by the sample; and providing narrow-band resonance amplification providing gain necessary to support self-oscillations in an opto-electronic loop comprising the light source, the sample, light directing apparatus, light receiving apparatus, and resonance amplification apparatus. In the preferred embodiment, the light source and the fluorescence light have different wavelengths and each carry a radio frequency intensity modulation in the loop. The light source can be a light emitting diode (e.g., blue). In the receiving step, a long-wavelength-pass optical filter is preferably employed to prevent reception of light from the light source. The directing step preferably directs light to a plurality of fluorophores, and the receiving step receives light with radio frequency intensity modulation of identical frequency as that of the light source but phase shifted. Receiving is preferably done by photodetector, such as a photomultiplier tube. In the receiving and providing steps, the photodetector is preferably connected to an input of the resonance amplification apparatus, which is preferably a resonance radio frequency amplifier, most preferably with a central frequency of the amplifier and lengths of the light directing and receiving apparatus being such that an RF modulation frequency multiplied by an excited-state fluorescence lifetime of fluorophores in the sample is approximately one. A frequency counter is preferably employed to receive a signal between the amplifier and the light source, together with a second light source and a photodetector receiving output from the second light source and providing input to the frequency counter. The light source may be a diode laser (e.g., blue or red). The resonance amplification preferably prevents higher order oscillations. In an alternative embodiment, an electronic phase shifter is employed within the opto-electronic loop and a second opto-electronic loop is employed comprising the electronic phase shifter, a second apparatus for receiving fluorescence light generated by the sample, and a phase detector. The method preferably provides a sub-picosecond resolution for changes in average excited-state lifetime of the sample. The method can measure changes of chemical environment when the sample exhibits changes of fluorescence average excited-state lifetime in response to changes of the chemical environment, changes of physical environment when the sample exhibits changes of fluorescence average excited-state lifetime in response to changes of the physical environment, changes of concentration of one or more chemical species when the sample exhibits changes of fluorescence average excited-state lifetime in response to changes of concentration of the chemical species, and changes of concentration of one or more biological species when the sample exhibits changes of fluorescence average excited-state lifetime in response to changes of concentration of the biological species. The method can obtain directly information regarding changes of concentration in a species selected from the group consisting of chemical and biological species from measurement of self-oscillation frequency in the opto-electronic loop.
The invention is further of a fluorescence average absolute lifetime sensing method comprising: exciting a fluorescence excitation light source; directing light from the light source to a sample; receiving fluorescence light generated by the sample; employing an electronic phase shifter; and providing narrow-band resonance amplification providing gain necessary to support self-oscillations in an opto-electronic loop comprising the light source, the sample, light directing apparatus, light receiving apparatus, the phase shifter, and resonance amplification apparatus. In the preferred embodiment, the providing step employs a resonance radio frequency amplifier, preferably with a central frequency of the amplifier and lengths of the light directing and receiving apparatus being such that an RF modulation frequency multiplied by an excited-state fluorescence lifetime of fluorophores in the sample is not approximately one. The directing step preferably directs light to a plurality of fluorophores, with the receiving step receiving light with radio frequency intensity modulation of identical frequency as that of the light source but phase shifted.